Process for producing beta-glucanase by foam separation coupled fermentation

Abstract

A process for producing beta-glucanase by coupling foam separation and fermentation relates to the technical field of foam separation and comprises the following steps: 1) pretreatment; (2) assembling an instrument; 3) system debugging; 4) performing high-pressure steam sterilization; 5) precise inoculation; (6) fermenting and supplementing materials; and 7) extraction and detection. According to the method disclosed by the invention, efficient production of beta-glucanase taking pichia pastoris as a fermentation main body is successfully realized, and the foam separation technology is introduced, so that the production cost is remarkably reduced, the operation process is simplified, and the yield and the enzyme activity of the product are greatly improved. The method has the remarkable advantages of being low in cost, easy to operate, high in yield and high in enzyme activity, and a new thought is provided for industrial production of beta-glucanase.

Description

Technical Field

[0001] The invention relates to the technical field of foam separation, in particular to a process for producing beta-glucanase by foam separation coupled with fermentation. Background Art

[0002] β-glucanase is a type of enzyme that can hydrolyze β-glucan and is widely used in the food industry, feed industry, pharmaceutical field and environmental protection field. Traditional production methods of β-glucanase include solid culture, immobilized cell fermentation, liquid culture, etc. However, solid culture has the disadvantages of difficult control of production parameters, low production efficiency, uneven fermentation, extensive process, and large equipment footprint; the cell immobilization method may have side reactions and it is difficult to ensure the purity of the obtained product and the enzyme activity is easily affected. Although the liquid deep fermentation method has a short fermentation cycle, it has the disadvantages of not being able to separate the product from the fermentation system in time, and the product accumulation causes product inhibition, thereby affecting production efficiency. Summary of the invention

[0003] The purpose of the present invention is to solve the above-mentioned problems in the prior art and provide a process for producing β-glucanase by foam separation coupled with fermentation, which cleverly combines the foam separation technology with the fermentation process, greatly reduces the production cost while maintaining high production efficiency, and makes the industrial production of β-glucanase more economical and feasible.

[0004] In order to achieve the above object, the present invention adopts the following technical scheme:

[0005] A process for producing β-glucanase by foam separation coupled with fermentation, comprising the following steps:

[0006] 1) Pretreatment: Activate and cultivate the bacteria, and screen, immerse in ethanol and remove residues from the macroporous adsorption resin;

[0007] 2) Device assembly: Assemble a foam separation coupled fermentation device, including a fermentation tank and a foam collection tank, wherein the fermentation tank is filled with culture medium, the foam collection tank is filled with pretreated macroporous resin, and the fermentation tank and the foam collection tank are connected by a pipeline;

[0008] 3) Sterilization and inoculation: After sterilizing the foam separation coupled fermentation device with high pressure steam, the activated bacteria in step 1) are inoculated into the fermentation tank;

[0009] 4) Dynamic feeding and induced enzyme production: Glycerol and methanol-sorbitol mixed carbon sources are added in stages during the fermentation process, and enzyme production is induced by adjusting the temperature;

[0010] 5) Product separation and detection: The β-glucanase is transferred to a collection tank along with the foam using foam separation technology. The products are extracted from the fermentation broth, foam liquid and resin, and the enzyme yield and activity are measured.

[0011] The bacterial strain in step 1) is recombinant Pichia pastoris GS115 / pPIC9K-bgl.

[0012] In step 2), the foam separation coupled fermentation device includes the fermentation tank and foam collection tank, a foam separation tube, a temperature control system, a pH electrode, and a safety bottle; the fermentation tank and the foam collection tank are connected via the foam separation tube, the safety bottle is connected to the foam collection tank, the temperature control system is used to control the temperature of the fermentation tank, and the pH electrode is used to detect the pH of the fermentation tank.

[0013] In step 2), the culture medium is BSM culture medium, and the salt concentration of the BSM culture medium is 0.2-1 of the original concentration.

[0014] More preferably, the salt concentration of the BSM medium in step 2) is 0.7 of the original concentration.

[0015] In step 3), the temperature of the fermentation tank is 25°C to 35°C during inoculation, and the volume of the inoculated seed culture solution is 5% to 15% of the culture medium.

[0016] The feeding parameters in step 4) are as follows: glycerol feeding stage: add glycerol containing PTM1 after 20-28 h of fermentation and continue for 10-14 h; methanol-sorbitol mixed carbon source feeding stage: stop glycerol feeding for 0.5-1.5 h, then add a mixed carbon source with a methanol mass ratio of 0.8-0.95 at a rate of 1-1.4 mL / h and continue for 96-144 h.

[0017] More preferably, the feeding rate of the methanol-sorbitol mixed carbon source is 1.4 mL / h.

[0018] More preferably, in the mixed carbon source in step 4), the methanol accounts for 0.85% by mass.

[0019] In step 4), the fermentation temperature is 23-27° C. during the mixed carbon source feeding stage. Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0020] The process of the present invention can not only effectively relieve product inhibition and improve production efficiency, but also significantly reduce production costs by coupling foam separation technology during the fermentation process. By optimizing fermentation parameters, including feed rate, mixed carbon source ratio, and BSM medium salt concentration, the process can achieve efficient production of β-glucanase.

[0021] The present invention uses Pichia pastoris as the fermentation strain, and investigates the effects of the ratio of methanol-sorbitol mixed carbon source, feed rate and salt concentration of BSM medium on the fermentation effect through single factor exploration experiments; optimizes the fermentation production conditions through orthogonal experiments; and finally verifies it through tank fermentation experiments. The results show that the optimal fermentation parameter combination is a mixed carbon source ratio (methanol mass ratio) of 0.85, a feed rate of 1.4 mL / h, and a BSM salt concentration of 70% of the original BSM salt concentration. At this time, the enzyme production and enzyme activity of β-glucanase are 1292 mg / L and 2171 U / mL, respectively, which also confirms the feasibility of using methanol-sorbitol mixed carbon source for feeding to make up for biomass loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of a foam separation device; wherein, it comprises a foam separation tube 1, a gas outlet 2, a safety bottle 3, a foam collection tank 4, a temperature sensor 5, a cooling plate 6, a temperature control system 7, a heating jacket 8, a fermentation tank 9, a pH electrode 10, and a fixing bracket 11.

[0023] Figure 2 This is a comparison chart of β-glucanase production in the control experiment.

[0024] Figure 3 This is a comparison chart of cell dry weight in the control experiment.

[0025] Figure 4 This is a comparison chart of enzyme activity in the control experiment.

[0026] Figure 5 This is a comparison chart of the total production of β-glucanase in the control experiment.

[0027] Figure 6 This is a graph showing the effect of feed rate on cell dry weight.

[0028] Figure 7 This is a curve chart showing the effect of feed rate on β-glucanase production.

[0029] Figure 8 This is a curve diagram showing the effect of feed rate on enzyme activity.

[0030] Fig. 9 This is a curve chart showing the effect of the ratio of mixed carbon sources on cell dry weight.

[0031] Fig.10 This is a curve chart showing the effect of the mixed carbon source ratio on β-glucanase production.

[0032] Fig.11 This is a curve chart showing the effect of the ratio of mixed carbon sources on the activity of β-glucanase.

[0033] Fig.12 This is a curve chart showing the effect of BSM salt concentration on cell dry weight.

[0034] Fig.13 This is a curve chart showing the effect of BSM salt concentration on enzyme amount.

[0035] Fig.14 This is a curve chart showing the effect of BSM salt concentration on enzyme activity.

[0036] Fig.15 The orthogonal experiment results are analyzed; (a) K value distribution of enzyme amount; (b) R value distribution of enzyme amount; (c) K′ value distribution of enzyme activity; (d) R′ value distribution of enzyme activity.

[0037] Fig.16 Comparison chart of β-glucanase production in the verification experiment.

[0038] Fig.17 Comparison chart of cell dry weight for verification experiment.

[0039] Fig.18 Comparison chart of enzyme activity for verification experiment.

[0040] Fig.19 Comparison chart of total β-glucanase production in the validation experiment. DETAILED DESCRIPTION

[0041] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and understandable, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the scope of the present invention is not limited by these embodiments. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the following embodiments are only used to illustrate the principles of the present invention, and the various processes and methods not described in detail are conventional methods known in the art. It should be understood that these embodiments are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way.

[0042] See also Figure 1 The foam separation coupled fermentation device of the present invention comprises a foam separation pipe 1, a gas outlet 2, a safety bottle 3, a foam collection tank 4, a temperature sensor 5, a cooling plate 6, a temperature control system 7, a heating jacket 8, a fermentation tank 9, a pH electrode 10, and a fixing bracket 11;

[0043] Wherein, flange interfaces are provided at both ends of the foam separation tube 1, which are respectively connected to the fermentation tank 9 and the foam collection tank 4 through a fixed bracket 11. Specifically, a stainless steel cover plate is installed at the bottle mouth of the foam collection tank 4, which is connected to the foam separation tube through a silicone hose (with a screen inside); the safety bottle 3 is connected to the foam collection tank 4 to form an overflow channel. When the amount of foam is too large, the excess foam flows into the safety bottle and is discharged from the air outlet to avoid excessive system pressure; the probe thermal resistor of the temperature sensor 5 is directly inserted into the fermentation tank 9 to monitor the temperature in real time, and is connected to the temperature control system 7, and the feedback signal is used to regulate the heating jacket 8 and the cooling plate 6; the heating jacket 8 tightly wraps the periphery of the fermentation tank 9; The cooling plate 6 is attached to the bottom of the fermentation tank 9 and connected to the low-temperature water tank through a silicone tube, and together with the heating jacket 8, the temperature of the fermentation liquid is precisely controlled; the temperature control system 7 is connected to the heating jacket and the cooling circulating water device through a circuit, and automatically adjusts the power according to the temperature sensor signal; a threaded cover (including a feed port, an air inlet, a sampling port, and a pH electrode interface) is provided on the top of the fermentation tank 9, which is connected to the fixed bracket through a stainless steel cover plate, and an air filter membrane is provided at the air inlet; the pH electrode 10 is inserted into the fermentation tank through a dedicated interface to monitor the pH value in real time; the fixed bracket 11 integrates and fixes the fermentation tank, foam separation tube, foam collection tank and other components through screws and nuts to ensure overall stability.

[0044] Example 1

[0045] A process for producing β-glucanase by foam separation coupled with fermentation, the steps are as follows:

[0046] S1. Pretreatment: Pretreat the macroporous adsorption resin in advance, and activate and culture the bacteria.

[0047] Weigh an appropriate amount of macroporous adsorption resin using an electronic balance, prepare a 40-mesh screen to filter it, and retain the resin with a particle size larger than 40 mesh; rinse the sieved resin repeatedly with anhydrous ethanol several times and soak it in a container for 24 hours; take out the resin after soaking and rinse it repeatedly with distilled water until the alcohol taste remaining on the resin is removed. Activate and culture the recombinant Pichia GS115 / pPIC9K-bgl.

[0048] S2. Instrument assembly: Assemble the foam separation coupled fermentation device. The assembled foam separation coupled fermentation instrument is as shown in the attached Figure 1 600 mL of BSM medium was placed in the fermentation tank, and the pretreated macroporous resin was placed in the foam collection tank. Then two magnetic beads were placed in the fermentation tank and the foam collection tank respectively. The preparation method of BSM medium was as follows: 40 g of glycerol, 26.7 mL of 85% (m / m) H 3 PO 4 , 0.93 g CaSO 4 ·2H 2O, 18.2 g K 2 SO 4 、14.9 g MgSO 4 7H 2 O, 4.13 g KOH, adjust the pH to 5.0 with 28% ammonia water, sterilize at 121 °C and add 2 mL PTM1.

[0049] S3. System debugging: Check whether the feeding and ventilation pipelines are damaged, connect the pH electrode, temperature sensor and various pipelines, and calibrate the pH electrode.

[0050] S4. High-pressure steam sterilization: Seal the tops of the fermentation tank and foam collection tank with sterile breathable sealing films, clamp the ends of each pipe and air filter membrane with water-stop clamps, and wrap the pH electrode, air filter membrane, and sampling port with tin foil. Place the processed foam separation coupling fermentation device into a high-pressure steam sterilizer for sterilization, ensuring that all parts and culture media are sterile. Remove the water-stop clamps and tin foil after sterilization.

[0051] S5. Precise inoculation: Turn on the magnetic stirrer to stir and ensure that the culture medium is evenly mixed. Turn on the air compressor to let in air to provide oxygen for the fermentation process. Connect the cooling circulating water, cool it to the appropriate fermentation temperature of 30°C, and inoculate the seed culture solution with a volume of 10% of the culture medium.

[0052] S6. Fermentation and feeding: 24 h after the start of fermentation, 50% glycerol (containing 12 mL / L of PTM1) was fed at a feeding rate of 2.4 mL / h to provide additional carbon source and nutrients for the fermentation process; 12 h after the addition of glycerol was stopped, the fermentation temperature was adjusted to 25 °C; 1 h after the cessation of the addition of glycerol, methanol was fed at a feeding rate of 1.2 mL / h to induce enzyme production; 120 h after the start of the addition of carbon source, the fermentation was terminated and the fermentation tank was cleaned to complete the entire fermentation process.

[0053] S7. Extraction and detection: After the foam separation coupled fermentation is completed, the produced β-glucanase is distributed in the fermentation liquid, foam liquid and resin, which are extracted and detected respectively to obtain the cell dry weight, yield and enzyme activity of the β-glucanase.

[0054] Example 2

[0055] Compared with Example 1, the difference between this embodiment and Example 1 is that a traditional fermentation tank is used to produce β-glucanase, and foam separation coupled fermentation technology is not used, and only the solution in the fermentation tank is extracted and detected.

[0056] Example 3

[0057] In Example 1, the feeding rate of the carbon source in step S6 is 1.2 mL / h; the difference between this example and Example 1 is that the feeding rates are 1.0, 1.1, 1.3, and 1.4 mL / h, respectively.

[0058] Example 4

[0059] In Example 1, the carbon source added in step S6 is methanol; the difference between this example and Example 1 is that the added carbon source is a methanol-sorbitol mixed carbon source, and the mass proportions of methanol are 0.80, 0.85, 0.90, and 0.95, respectively.

[0060] Example 5

[0061] In Example 1, the BSM salt concentration in step S2 is the original salt concentration; the difference between this example and Example 1 is that the BSM salt concentration is 0.2, 0.4, 0.6, and 0.8 of the original BSM salt concentration.

[0062] Example 6

[0063] The difference between this embodiment and embodiment 1 is that:

[0064] Carbon source feed rate and mixed carbon source ratio in step S6, BSM salt concentration in step S4: Four levels were selected for each parameter based on the single factor experimental results of Example 1 and Examples 3, 4, and 5, and a three-factor three-level L 9 (3 4 ) Design an orthogonal experiment, and the factor levels are shown in Table 1.

[0065] Table 1 Factor levels of orthogonal experiment for foam separation coupled with fermentation to produce β-glucanase

[0066]

[0067] Example 7

[0068] The difference between this embodiment and embodiment 1 is that:

[0069] The carbon source feeding rate and mixed carbon source ratio of step S6, and the BSM salt concentration of step S4: the optimal fermentation parameter combination of the orthogonal experiment in Example 6 is a mixed carbon source ratio (methanol mass percentage) of 0.85, a feeding rate of 1.4 mL / h, and a BSM salt concentration of 70% of the original BSM salt concentration.

[0070] Example 8

[0071] The difference between this embodiment and embodiment 2 is that:

[0072] The carbon source feeding rate and mixed carbon source ratio of step S6, and the BSM salt concentration of step S4: the optimal fermentation parameter combination of the orthogonal experiment in Example 6 is a mixed carbon source ratio (methanol mass percentage) of 0.85, a feeding rate of 1.4 mL / h, and a BSM salt concentration of 70% of the original BSM salt concentration.

[0073] Index determination

[0074] Determination of dry weight of bacteria: The dry weight of bacteria was determined by centrifugal drying. Take an appropriate amount of 1.5 mL empty centrifuge tubes, weigh and number them, and record the weight of the empty tubes. Pipette 1 mL of fermentation liquid into the corresponding centrifuge tubes, set the speed to 10,000 rpm for 5 min, pour out the supernatant, and then invert to drain excess water. Put the centrifuge tube containing wet bacteria into the oven to dry the moisture, weigh and record the weight of the tube after drying until constant weight. Dry weight of bacteria = total weight of the centrifuge tube after drying - weight of the empty centrifuge tube.

[0075] Determination of β-glucanase concentration: The β-glucanase concentration was determined by Coomassie brilliant blue staining. The sample solution was centrifuged at low temperature for 10 min, and 800 μL of sterile distilled water and 200 μL of supernatant were transferred to a test tube. 5 mL of Coomassie brilliant blue dye solution was added, shaken well, and allowed to stand for 5 min. A test tube with 1 mL of sterile distilled water was used as a blank control, the absorbance was measured at 595 nm, and the β-glucanase concentration was found from the standard curve.

[0076] Determination of β-glucanase activity: The enzyme activity is determined by the color reaction between the hydrolysis product of β-glucanase and the DNS reagent. First, take 1 mL of bacterial solution and centrifuge it at 10,000 rpm for 5 min. Pour the supernatant into a new 1.5 mL centrifuge tube, precipitate and lyse the cells. After lysis, obtain the enzyme sample to be tested. Take 1 mL of substrate solution and place it in four test tubes, three of which are test tubes and one is a blank control. Keep it warm at 50±0.2℃ for 2~3 min. After the insulation is completed, add 1 mL of enzyme solution to each of the three test tubes, and add 1 mL of distilled water to the blank tube as a control. Then heat the four test tubes in a 50℃ water bath for 10 min at the same time. After taking them out, add 2 mL of DNS reagent to each tube and heat them in boiling water for 5 min. After cooling, add 10 mL of deionized water to each tube and mix well. Finally, adjust the blank tube to zero and measure the absorbance at 540 nm. The absorbance of each parallel sample was averaged and the β-glucanase activity was calculated according to the following formula:

[0077]

[0078] Wherein, x is the average OD value of the sample; b and a are obtained by regression equation based on maltose concentration and corresponding OD value; n is the dilution multiple of enzyme solution; 10 is the time of enzymatic reaction in minutes; F is substrate correction factor (1.047); W is the weight of enzyme sample (1 mL);

[0079] Test Result 1

[0080] Example 1 and Example 2 are feasibility studies on the production of β-glucanase by foam separation coupled with fermentation. Example 1 is the experimental group and Example 2 is the control group. The test results are as follows:

[0081] like Figure 2 As shown in the figure, after 96 h of fermentation, the amount of enzyme in the fermentation broth of the experimental group continued to be higher than that of the control group. This is because the accumulation of fermentation products in the control group was more inhibited by the product, resulting in a slower growth rate of the bacteria and a slower fermentation rate.

[0082] like Figure 3 As shown in the figure, from 0 to 84 h after fermentation, the cell dry weight of the control group was greater than that of the experimental group. This is because in the middle of the fermentation, a large amount of foam was produced, and some of the bacteria in the fermentation liquid were taken away by the foam and entered the collection tank. Even if the fermentation device was equipped with a circulation reflux device, there would inevitably be a certain loss of biomass, resulting in a decrease in cell dry weight. In addition, during the fermentation of the experimental group, the product was continuously removed from the fermentation tank along with the foam liquid, which reduced the product inhibition. In this case, Pichia pastoris could use more methanol to induce the production of β-glucanase rather than increase biomass. Afterwards, with the reflux of the foam liquid and continuous feeding, the cell dry weight continued to increase.

[0083] like Figure 4 As shown in the figure, after 60 h of fermentation, the product enzyme activity in the foam separation coupled fermentation process was always greater than that in the non-coupled fermentation process, and there was a large gap in the maximum enzyme activity, which reflected the superiority of foam separation coupled fermentation.

[0084] like Figure 5 As shown, the total production of β-glucanase is the sum of all β-glucanases in the fermentation broth, foam liquid and macroporous resin. The total enzyme production of the experimental group is significantly higher than that of the control group. The calculation shows that the increase in β-glucanase production is 33%.

[0085] Test Result 2

[0086] Example 1 and Example 3 are single factor experiments on feeding rate, and the test results are listed as follows:

[0087] like Figures 6 to 8As shown in the figure, as the feed rate increased from 1.0 mL / h to 1.4 mL / h, the dry weight of the bacteria, β-glucanase, and β-glucanase activity all showed a general trend of first increasing and then decreasing. When the carbon source supplementation rate was 1.3 mL / h, the dry weight of the bacteria and the β-glucanase activity reached the maximum values ​​of 17.75 g / L and 803.39 U / mL, respectively; when the feed rate was 1.2 mL / h, the β-glucanase production reached the maximum value of 1094.06 mg / L. Since the difference between the β-glucanase production at the feed rate of 1.3 mL / h and that at the feed rate of 1.2 mL / h was not large, and as shown in the figure, Figure 8 The enzymatic activity of β-glucanase at a feed rate of 1.3 mL / h was significantly different from that at 1.2 mL / h, so a feed rate of 1.3 mL / h was determined as the optimal feed rate condition for subsequent experiments.

[0088] Test Result 3

[0089] Example 1 and Example 4 are single factor experiments on the ratio of mixed carbon sources, and the test results are listed as follows:

[0090] like Figures 9 to 11 As shown in the figure, as the mixed carbon source ratio increases from 0.80 to 1.00, the β-glucanase production and the dry weight of the bacteria show a trend of first increasing and then decreasing with the increase of the mass proportion of methanol in the mixed carbon source, and the enzyme activity shows a trend of first decreasing and then increasing. This is due to the insufficient amount of methanol feed, the weakening of the inducing enzyme production, and the excessively high methanol concentration will inhibit the growth of bacteria. When the mixed carbon source ratio is 0.90, the β-glucanase production reaches a maximum value of 1140.32 mg / L. After comprehensive consideration, the mixed carbon source ratio (methanol mass proportion) of 0.9 was selected as the optimal mixed carbon source ratio single factor experimental result for subsequent experiments.

[0091] Test Result 4

[0092] Example 1 and Example 5 are single factor experiments of BSM salt concentration, and the test results are listed as follows:

[0093] like Fig.12 and Fig.13 As shown in the figure, as the BSM salt concentration increased from 0.2 to 1.0, the cell dry weight and β-glucanase production showed a trend of first increasing and then decreasing, and both reached their maximum values ​​at the original BSM salt concentration of 0.8, which were 18.00 g / L and 1292.56 mg / L, respectively. Fig.14As shown in the figure, as the BSM salt concentration increased from 0.2 of the original BSM salt concentration to 1.0, the enzyme activity showed a trend of first decreasing and then increasing, reaching the minimum value at 0.8 of the original BSM salt concentration. However, considering that the BSM salt concentration has a greater impact on the cell dry weight and β-glucanase production, it was determined that the BSM salt concentration of 0.8 of the original concentration was the optimal, and this condition was used for subsequent experiments.

[0094] Test result 5

[0095] Example 6 was an orthogonal experiment, and the test results are listed as follows:

[0096] The specific experimental plans and result analyses of sixteen groups were obtained through the factor levels of the orthogonal table as shown in Table 2. Among them, K1, K2, K3, and K4 were the means of the enzyme production corresponding to the four levels of each single factor, and the R value was the range of each K value; K1′, K2′, K3′, and K4′ were the means of the enzyme activity corresponding to the four levels of each single factor, and the R′ value was the range of each K′ value.

[0097] Table 2 Specific experimental plans and result analyses of the orthogonal experiment

[0098]

[0099] As shown in Table 2 and Fig.15 shown, when using the β-glucanase production as the measurement standard, the highest enzyme production could be achieved when the mixed carbon source ratio, feeding rate, and BSM salt concentration were 0.85, 1.4 mL / h, and 0.85 respectively. The order of the influence degree of each factor on the enzyme production was: mixed carbon source ratio > feeding rate > BSM salt concentration; when using the β-glucanase activity as the measurement standard, the highest enzyme activity could be achieved when the mixed carbon source ratio, feeding rate, and BSM salt concentration were 0.85, 1.4 mL / h, and 0.7 respectively. The order of the influence degree of each factor on the enzyme activity was: mixed carbon source ratio < feeding rate < BSM salt concentration. Since the enzyme activity at a BSM salt concentration of 0.7 of the original concentration had an obvious advantage over that at 0.85, the optimal experimental conditions were determined as: mixed carbon source ratio (methanol mass ratio) 0.85, feeding rate 1.4 mL / h, and BSM salt concentration of 0.7 of the original BSM salt concentration. At this time, the enzyme production and enzyme activity of β-glucanase were 1292 mg / L and 2171 U / mL respectively.

[0100] Test result 6

[0101] Examples 7 and 8 were verification experiments of the orthogonal experiment results. Example 7 was the experimental group, and Example 8 was the control group. The test results are listed as follows:

[0102] As Fig.16As shown in the figure, the enzyme production of the control group was significantly higher than that of the experimental group at 0-60 h after fermentation. However, after 72 h of fermentation, the product accumulation in the fermentation broth of the control group inhibited the fermentation process, and the enzyme content in the fermentation broth of the experimental group began to be higher than that of the control group and showed an increasing trend.

[0103] like Fig.17 As shown in the figure, the experiment adopted the method of supplementing with a mixed carbon source composed of methanol and sorbitol to compensate for the loss of biomass during the fermentation process, which was effective. There was no significant difference in the biomass between the control group and the experimental group in the early stage of fermentation. After 72 hours of fermentation, the growth of the bacteria in the fermentation broth of the control group gradually slowed down due to product inhibition. In contrast, the foam separation coupled fermentation technology used in the experimental group was able to promptly relieve the product inhibition phenomenon. With the reflux of the foam liquid and continuous supplementation, the dry weight of the bacteria continued to grow.

[0104] like Fig.18 As shown in the figure, after 48 h of fermentation, the enzyme activity of the experimental group was significantly higher than that of the control group and there was a large gap in the maximum enzyme activity, which reflected the superiority of foam separation coupled fermentation.

[0105] like Fig.19 As shown in the figure, the total production of β-glucanase in the experimental group is the sum of all β-glucanase in the fermentation broth, foam liquid and macroporous resin. It can be seen intuitively from the figure that the total enzyme production of the experimental group is significantly higher than that of the control group. After calculation, the increase in β-glucanase production is 47%.

[0106] By comparing the test results of Example 1 and Example 2, it is verified that the foam separation coupled fermentation can greatly increase the product yield while ensuring enzyme activity compared with the traditional tank fermentation. Compared with the traditional tank fermentation experiment, the product yield is increased by 33%.

[0107] By comparing the test results of Example 1 with those of Examples 3, 4, and 5, that is, considering the effects of single factors such as feed rate, methanol-sorbitol mixed carbon source ratio, and BSM salt concentration on the production of β-glucanase by foam separation coupled fermentation, the optimal fermentation parameters of the single factor experiment were determined to be: feed rate of 1.3 mL / (h·L fermentation liquid), mixed carbon source ratio (methanol mass proportion) of 0.9, and BSM salt concentration of 0.8 of the original salt concentration.

[0108] According to this result, Example 6 comprehensively considers the effects of three factors, namely, feed rate, mixed carbon source ratio and BSM salt concentration, on the production of β-glucanase by foam separation coupled fermentation, and determines that the optimal fermentation parameter combination of the orthogonal experiment is a mixed carbon source ratio (methanol mass ratio) of 0.85, a feed rate of 1.4 mL / h, and a BSM salt concentration of 70% of the original BSM salt concentration. At this time, the enzyme production and enzyme activity of β-glucanase are 1292 mg / L and 2171 U / mL, respectively. Since the single-factor experiment only considers a single factor, and the orthogonal experiment comprehensively considers multiple factors, the optimal conditions obtained are different.

[0109] By comparing the test results of Example 7 and Example 8, it was verified that under the optimal fermentation parameter combination of the orthogonal experiment, the enzyme production of the foam separation coupled fermentation technology increased by 47% compared with the traditional fermentation technology.

[0110] In summary, the present invention provides a low-cost, easy-to-operate, and high-efficiency process for producing β-glucanase by foam separation coupled with fermentation, which provides a new idea for the industrial production of β-glucanase.

Claims

1. A process for producing β-glucanase by foam separation coupled with fermentation, characterized in that: The following steps are involved: 1) Pretreatment: Activate and cultivate the bacteria, and screen, immerse in ethanol and remove residues from the macroporous adsorption resin; 2) Device assembly: Assemble a foam separation coupled fermentation device, including a fermentation tank and a foam collection tank, wherein the fermentation tank is filled with culture medium, the foam collection tank is filled with pretreated macroporous resin, and the fermentation tank and the foam collection tank are connected by a pipeline; 3) Sterilization and inoculation: After sterilizing the foam separation coupled fermentation device with high pressure steam, the activated bacteria in step 1) are inoculated into the fermentation tank; 4) Dynamic feeding and induced enzyme production: Glycerol and methanol-sorbitol mixed carbon sources are added in stages during the fermentation process, and enzyme production is induced by adjusting the temperature; 5) Product separation and detection: The β-glucanase is transferred to a collection tank along with the foam using foam separation technology. The products are extracted from the fermentation broth, foam liquid and resin, and the enzyme yield and activity are measured.

2. The process for producing β-glucanase by foam separation coupled with fermentation according to claim 1, characterized in that: In step 1), the bacterial strain is recombinant Pichia pastoris GS115 / pPIC9K-bgl.

3. The process for producing β-glucanase by foam separation coupled with fermentation according to claim 1, characterized in that: In step 2), the foam separation coupled fermentation device includes the fermentation tank and foam collection tank, a foam separation tube, a temperature control system, a pH electrode, and a safety bottle; the fermentation tank and the foam collection tank are connected via the foam separation tube, the safety bottle is connected to the foam collection tank, the temperature control system is used to control the temperature of the fermentation tank, and the pH electrode is used to detect the pH of the fermentation tank.

4. The process for producing β-glucanase by foam separation coupled with fermentation according to claim 1, characterized in that: In step 2), the culture medium is BSM culture medium, and the salt concentration of BSM culture medium is 0.2-1 of the original concentration.

5. The process for producing β-glucanase by foam separation coupled with fermentation according to claim 4, characterized in that: The salt concentration of the BSM medium was 0.7 of the original concentration.

6. The process for producing β-glucanase by foam separation coupled with fermentation according to claim 1, characterized in that: In step 3), the temperature of the fermentation tank is 25°C to 35°C during inoculation, and the volume of the inoculated seed culture solution is 5% to 15% of the culture medium.

7. The process for producing β-glucanase by foam separation coupled with fermentation according to claim 1, characterized in that: The feeding parameters in step 4) are as follows: glycerol feeding stage: add glycerol containing PTM1 after 20-28 h of fermentation and continue for 10-14 h; methanol-sorbitol mixed carbon source feeding stage: stop glycerol feeding for 0.5-1.5 h, then add a mixed carbon source with a methanol mass ratio of 0.8-0.95 at a rate of 1-1.4 mL / h and continue for 96-144 h.

8. The process for producing β-glucanase by foam separation coupled with fermentation according to claim 7, characterized in that: The feeding rate of the methanol-sorbitol mixed carbon source is 1.4 mL / h.

9. The process for producing β-glucanase by foam separation coupled with fermentation according to claim 7, characterized in that: In the mixed carbon source, the methanol accounts for 0.85% by mass.

10. The process for producing β-glucanase by foam separation coupled with fermentation according to claim 7, characterized in that: The fermentation temperature was 23~27℃ during the mixed carbon source feeding stage.

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